CN114651723A - Culture medium and method for inducing one-step seedling formation of rhododendron pseudobulb plant - Google Patents
Culture medium and method for inducing one-step seedling formation of rhododendron pseudobulb plant Download PDFInfo
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Abstract
The invention discloses a method for inducing plants to grow seedlings in one step by using pseudobulbs of rhododendron and application thereof, comprising the following steps: explant selection and cleaning: selecting underground pseudobulb, washing with saturated soap water and tap water, scraping off the surface bulges and depressions, adding tween-80, shaking, and spraying with tap water; explant disinfection: sequentially sterilizing with alcohol, saturated bleaching powder, benzalkonium bromide and mercuric chloride solution; and (3) induction culture of protocorms: cutting into small pieces with epidermis, and inoculating in protocorm induction culture medium; one-step seedling culture: cutting protocorm into small pieces, inoculating in one-step seedling culture medium, and culturing. The protocorm can directly obtain a complete plant through one-step seedling culture to form a commercial seedling, and the differentiation rate of the commercial seedling into a plant is 100 percent; during differentiation, the plant also takes root and forms pseudo bulbs, the rooting rate can reach 98 percent, and the pseudo bulb forming rate can reach 92 percent; high differentiation rate and rooting rate, greatly saved production cost, high pseudobulb forming rate and greatly improved domestication survival rate.
Description
Technical Field
The invention relates to the field of medicinal plant tissue culture and traditional Chinese medicinal materials, in particular to a method for inducing plants to grow seedlings in one step by using pseudobulbs of rhododendron simsii and application thereof.
Background
The dry pseudobulb of the azalea orchid is used as a medicine, the name of the medicine is pleione bulb, the medicine is one of the basic source plants of the pleione bulb recorded in Chinese pharmacopoeia at present, and the medicine is the pleione bulb medicine recorded in the herbal medicine of the past generation and widely recognized by modern scholars. Pseudobulbus Cremastrae Seu pleiones has effects of clearing heat and detoxicating, eliminating phlegm and resolving masses, and is mainly used for treating carbuncle, furunculosis, scrofula, subcutaneous nodule, snake and insect bite, and cripple. Modern medical research shows that the pleione contains phenanthrene compounds such as 7-hydroxy-2, 4-dimethoxyphenanthrene and the like, bibenzyl compounds such as 3, 5, 3' -trihydroxybibenzyl and the like, aromatic compounds such as cinnamic acid, protocatechuic acid and the like and glycosides thereof, sugar and glycoside compounds such as beta-daucosterol VIII, gastrodin, daucosterol and the like, terpenes and steroid compounds such as p-hydroxybenzoic acid, stigmasterol and the like and other compounds such as physcion, quercetin and the like, has the effects of resisting malignant tumors such as liver cancer, lung cancer, breast cancer and the like, reducing blood pressure, resisting bacteria, resisting oxidation, resisting ventilation, reducing blood fat, resisting atherosclerosis and the like, and the pleione can be clinically used for treating diseases such as internal, external, women, children, five sense organs, skin and the like, and the adaptation diseases are continuously expanded. In particular, the edible tulip is used as a compound medicine for clinical antitumor treatment and achieves good effect. At present, nearly 20 Chinese medicinal preparations taking the edible tulip as a raw material medicament mainly comprise Aiyu capsules, Xiaoru san Jie capsules, Zijin tablets and the like, wherein the Aiyu capsules are sold in the market at the maximum, and are mainly used for the auxiliary treatment of middle and late-stage cancers and the deficiency of qi and blood caused by leukopenia due to radiotherapy and chemotherapy of the cancers.
The rhododendron cannot be propagated sexually under natural conditions, although some units use the separated symbiotic bacteria to promote germination and have a certain promotion effect, the germination rate is still low, so that the conventional cultivation and planting mode mostly adopts an asexual propagation mode of digging wild resources and then carrying out plant division by using pseudobulbs to obtain seedlings, but the defects of long propagation period, low propagation rate and the like exist.
As the Cremastra Appendiculata seedlings are difficult to obtain in a large scale under natural conditions, with the continuous development of biotechnology, tissue culture technical researches on Cremastra Appendiculata are developed in various enterprises and public institutions in recent years. Most tissue culture studies use seeds as explants to obtain protocorms, and then obtain seedlings by proliferation, differentiation and rooting of the protocorms. On the one hand, due to the many original producing areas and the many varieties of the rhododendron, the drug effect and the single plant biological yield of the rhododendron are different in each producing area and variety; meanwhile, seeds are obtained by hybridization, so that genetic instability can be generated during the breeding of screened high-quality varieties, and the high-quality varieties cannot be stably inherited; on the other hand, the existing tissue culture technology needs to germinate seeds into protocorms, the protocorms are differentiated into seedlings with buds, leaves and other organs after being proliferated, and the seedlings take roots again, so that the process is complex; moreover, under the condition of the prior art, the differentiation rate is low, so that the large-scale production during tissue culture is greatly limited and the production cost is increased.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a one-step seedling method which has simplified production steps and high multiplication coefficient and can directly obtain a large number of rooted seedlings from the induced protocorm.
In order to achieve the purpose, the invention adopts the technical scheme that: a culture medium for inducing the one-step seedling formation of a rhododendron pseudobulb plant, wherein the culture medium comprises a protocorm induction culture medium and a one-step seedling formation culture medium,
the formula of the protocorm induction culture medium is as follows: n is a radical of6+2, 4-D0.3-0.5 mg/L +2-IP 1.0-1.2 mg/L + BR 0.05-0.1 mg/L + coconut milk 20-30 g/L + active carbon 0.5 g/L;
the formula of the one-step seedling culture medium is as follows: improvement B5+ NAA 0.5-0.6 mg/L + IBA 1.0-1.2 mg/L + TDZ 0.5-0.8 mg/L + BR 0.1-0.13 mg/L + hydrolyzed complexing protein (CH) 50-70 mg/L + coconut juice 40-50 ml/L + active carbon 0.5 g/L.
Further, the improvement B5The culture medium is original B5On the basis of the culture medium, the concentration of potassium nitrate is changed to 1500mg/L, the concentration of ammonium sulfate is changed to 205mg/L, the concentration of calcium chloride is changed to 125mg/L, the concentration of nicotinic acid is changed to 1.5mg/L, the concentration of pyridoxine hydrochloride is changed to 1.8mg/L, the concentration of zinc sulfate is changed to 3.0mg/L, and the rest elements are not changed.
The method for inducing the rhododendron pseudobulb plant to sprout in one step by using the culture medium comprises the following steps:
(1) explant selection and cleaning: selecting cremastra appendiculata, transplanting the cremastra appendiculata to a room, filling roots with 500 times of 75% carbendazim every 3 days for one time, continuously filling the roots for 4-5 times, after the root filling by the carbendazim is finished, taking underground pseudobulbs, removing overground stems, leaves and surface roots, rinsing with saturated soap water solution for 10-15 min, rinsing with tap water for 3-4 times, scraping the convex and concave parts of the surface by a knife, adding 2-3 drops of Tween-80, shaking for 10-15 min, and rinsing with the tap water for 60-80 min;
(2) explant disinfection: cleaning the explant, transferring the explant to a super clean workbench, disinfecting the explant with 75% (v/v) alcohol for 20-25 s, rinsing with sterile water for 2-3 times, disinfecting with saturated bleaching powder solution for 18-20 min, rinsing with sterile water for 3-4 times, disinfecting with 0.5% benzalkonium bromide solution for 20-25 min, rinsing with sterile water for 3-4 times, disinfecting with 0.08% (v/v) mercuric chloride solution for 6-7 min, and rinsing with sterile water for 6-8 times;
(3) and (3) induction culture of protocorms: the sterilized explants are cut into about 1.0cm after surface moisture is absorbed by sterile paper3Having epidermal bits, inoculated in N6+2, 4-D0.3-0.5 mg/L +2-IP 1.0-1.2 mg/L + BR 0.05-0.1 mg/L + coconut milk 20-30 g/L + active carbon 0.5g/L protocorm induction medium, culturing for 20 days under the condition of complete darkness at the temperature of 25 +/-2 ℃, and after 20 days, culturing for 30 days under the conditions of 25 +/-2 ℃, illumination intensity of 1000-2000 Lux and single day illumination for 10 hours in light-dark alternation;
(4) one-step seedling culture: inducing protocorm to cut into 1-1.5 cm, inoculating to modified B5+ NAA 0.5-0.6 mg/L + IBA 1.0-1.2 mg/L + TDZ 0.5-0.8 mg/L + BR 0.1-0.13 mg/L + CH 50-70 mg/L + coconut juice 40-50 ml/L + active carbon 0.5g/L in one-step seedling culture medium at 25 + -2 deg.CCulturing for 60-80 days in alternating dark at 2000-3000 Lux under the condition of 12h of daily illumination.
As a preferred embodiment of the present invention, the improvement B in the step (4)5The culture medium is original B5On the basis of the culture medium, the concentration of potassium nitrate is changed to 1500mg/L, ammonium sulfate is changed to 205mg/L, calcium chloride is changed to 125mg/L, nicotinic acid is changed to 1.5mg/L, pyridoxine hydrochloride is changed to 1.8mg/L, zinc sulfate is changed to 3.0mg/L, and other elements are not changed.
The method utilizes the rhododendron pseudobulb to induce the plant to grow seedlings in one step and is applied to the breeding of the rhododendron.
The beneficial technical effects of the invention are as follows:
1. the rhododendron has wide production area and various varieties, which causes great difference in quality of the rhododendron, and the rhododendron seeds are generally obtained by cross pollination of non-homologous plants, and the genetic characters of the rhododendron seeds can be changed by cross hybridization of the homologous plants during propagation of the high-quality rhododendron variety, so that the excellent characters of the rhododendron seeds cannot be well maintained. The invention adopts the pseudobulb as the explant, directly adopts the somatic cell of the female parent to breed, can completely keep the excellent characters of the female parent in the filial generation, and has extremely strong genetic stability.
2. The protocorm is cultured by a one-step seedling culture medium, so that a complete plant can be directly obtained to form a commercial seedling, the differentiation rate of the commercial seedling differentiated into the plant is 100%, the commercial seedling also roots and forms a pseudo bulb while differentiating, the rooting rate can reach 98%, and the pseudo bulb forming rate can reach 92%; high differentiation rate and rooting rate, greatly saved production cost, high pseudobulb forming rate and greatly improved domestication survival rate.
3. The method uses a one-step seedling culture medium, selects a certain concentration of NAA and IBA combination, adds a certain dose of TDZ, can make the protocorm have a certain differentiation and rooting, and simultaneously adds BR, hydrolyzed Casein (CH) and coconut juice, which is not only beneficial to improving the differentiation and rooting rate, but also can obtain a large amount of protocorms in the one-step seedling process, the protocorm can be continuously used for one-step seedling culture, and the multiplication coefficient can reach up to 16 in the seedling process within 60 days.
5. In the one-step seedling culture process, protocorms generated in the commercial seedling forming process can be domesticated in a field by the seedlings, pseudobulbs of the protocorms can be enlarged in the field domestication, the associated protocorms can obtain a large number of dragon eggs (commonly known as vegetative propagules of pleiones in a natural state), and the dragon eggs can be separated and cultured under certain conditions to form complete plants, so that further propagation is increased in the domestication process, and the propagation coefficient is further improved.
Drawings
FIG. 1 shows a commercial seedling produced by one-step seedling raising according to the method of the present invention;
FIG. 2 shows differentiation, rooting and proliferation of protocorm in one-step seedling culture;
FIG. 3 shows the initial growth stage of the protocorm in one-step seedling culture according to the method of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
Example 1
A method for inducing plants to sprout in one step by using pseudobulbs of rhododendron comprises the following steps:
1. explant selection and cleaning: selecting Cremastra appendiculata, transplanting to indoor, irrigating roots once every 3 days with 500 times of 75% (v/v) carbendazim solution, and irrigating roots 4 times continuously; after the carbendazim is filled into roots, taking underground pseudobulbs, removing overground stem leaves and surface root systems, rinsing for 10min by using a saturated soap water solution, rinsing for 3 times by using tap water, scraping the convex and concave parts of the epidermis by using a knife, adding 2 drops of Tween-80, shaking for 15min, and then flushing for 60min by using the tap water;
2. explant disinfection: transferring the cleaned explant to a super clean workbench, sterilizing the explant by using 75% (v/v) alcohol for 20s, rinsing the explant by using sterile water for 2 times, sterilizing the explant by using a saturated bleaching powder solution for 18min, rinsing the explant by using sterile water for 3 times, sterilizing the explant by using a 0.5% benzalkonium bromide solution for 20min, rinsing the explant by using the sterile water for 3 times, sterilizing the explant by using a 0.08% (v/v) mercuric chloride solution for 6min, rinsing the explant by using the sterile water for 6 times, wherein the sterilization pollution rate is 9.4%, and the sterilization mortality rate is 0;
3. and (3) induction culture of protocorms: the sterilized explants are cut into 1.0cm after absorbing surface water with sterile paper3Having epidermal pieces, inoculated in N6+2, 4-D0.3 mg/L +2-IP 1.0mg/L + BR 0.05mg/L + coconut milk 20g/L + active carbon 0.5g/L protocorm induction medium, culturing 20 days under the complete dark condition with the temperature of 25 +/-2 ℃, culturing 30 days under the conditions of 25 +/-2 ℃, the illumination intensity of 1000Lux and single day illumination of 10h after 20 days, wherein the protocorm induction rate is 68.3%;
4. one-step seedling culture: cutting the induced protocorm into 1cm, and inoculating to modified B5+ NAA 0.5mg/L + IBA 1.0mg/L + TDZ 0.5mg/L + BR 0.1mg/L + CH 50mg/L + coconut juice 40ml/L + activated carbon 0.5g/L, in the temperature of 25 + -2 deg.C, illumination intensity of 2000Lux, light and dark alternate culture under the condition of 12h of illumination light for 80 days, differentiation rate of 100%, rooting rate of 99.1%, pseudobulb forming rate of 91.7% (as shown in figure 1);
5. domestication: transplanting the Cremastra appendiculata tissue culture seedling obtained by one-step seedling formation into peat soil: perlite: in a matrix with coconut chaff at a ratio of 3:1:3, in a domestication environment under a greenhouse with a shading rate of 85%, keeping the air humidity at 100% for ten days before transplanting, then gradually reducing the air humidity, keeping the matrix humidity at 70-75% during the whole domestication period, simultaneously pouring 0.2% water-soluble compound fertilizer (15:15:15) every 15 days after domestication and transplanting for 30 days, domesticating for three months, wherein the domestication survival rate is 99.3%, the pseudobulb expands, and a dragon egg is formed at the bottom of the pseudobulb, and the formation rate of the dragon egg is 72.2%.
Example 2
A method for inducing plants to sprout in one step by using pseudobulbs of rhododendron comprises the following steps:
1. explant selection and cleaning: selecting Cremastra appendiculata, transplanting to indoor, irrigating root once every 3 days with 500 times of 75% (v/v) carbendazim solution, and irrigating root 5 times continuously; after the carbendazim is irrigated to the roots, underground pseudobulbs are taken, overground stem leaves and surface root systems are removed, the pseudobulbs are rinsed for 12min by using a saturated soap water solution, the pseudobulbs are rinsed for 4 times by using tap water, the convex and concave parts of the epidermis are scraped by using a knife, then 3 drops of tween-80 are added, the pseudobulbs are shaken for 13min, and then the pseudobulbs are flushed by using the tap water for 70 min;
2. explant disinfection: transferring the cleaned explant to a super clean workbench, disinfecting with 75% (v/v) alcohol for 25s, rinsing with sterile water for 3 times, disinfecting with saturated bleaching powder solution for 20min, rinsing with sterile water for 4 times, disinfecting with 0.5% neodell solution for 25min, rinsing with sterile water for 4 times, disinfecting with 0.08% (v/v) mercuric chloride solution for 6min, rinsing with sterile water for 7 times, wherein the disinfection pollution rate is 8.2%, and the disinfection death rate is 0.8%;
3. and (3) induction culture of protocorms: the sterilized explants are cut into about 1.0cm after absorbing surface moisture with sterile paper3Having epidermal bits, inoculated in N6+2, 4-D0.4 mg/L +2-IP 1.1mg/L + BR 0.8mg/L + coconut milk 30g/L + active carbon 0.5g/L protocorm induction medium, culturing 20 days under the complete dark condition with the temperature of 25 +/-2 ℃, after 20 days, culturing 30 days under the conditions of 25 +/-2 ℃, the illumination intensity of 2000Lux and single day illumination of 10h alternately in darkness, wherein the protocorm induction rate is 71.0%;
4. one-step seedling multiplication culture: cutting the induced protocorm into 1.5cm long, inoculating to modified B5+ NAA 0.6mg/L + IBA 1.1mg/L + TDZ 0.6mg/L + BR 0.12mg/L + CH 60mg/L + coconut juice 50g/L + active carbon 0.5g/L, in the temperature of 25 + -2 deg.C, illumination intensity of 3000Lux, light and dark alternate culture under the condition of sunshine light 12h for 80 days, differentiation rate is 100%, rooting rate is 97.3%, pseudobulb forming rate is 93.6%, proliferation coefficient is 17.2.
5. One-step seedling culture: taking out and domesticating the whole plant obtained by one-step seedling multiplication culture, cutting off the rest protocorm, cutting into 1.5cm long, and inoculating to the improved B5+ NAA 0.6mg/L + IBA 1.1mg/L + TDZ 0.6mg/L + BR 0.12mg/L + CH 60mg/L + coconut juice 50g/L + active carbon 0.5g/L, culturing alternately in dark and light conditions at 25 + -2 deg.C, illumination intensity 3000Lux and sunlight 12h for 70 days, differentiation rate of 100% and rooting rate of 97.8%Pseudobulb formation rate was 92.9% (as shown in fig. 2);
6. domestication: transplanting the rhododendron tissue culture seedlings obtained by one-step seedling formation into peat soil: perlite: in a matrix with coconut chaff at a ratio of 3:1:3, in a domestication environment under a greenhouse with a shading rate of 85%, keeping the air humidity at 100% for ten days before transplanting, then gradually reducing the air humidity, keeping the matrix humidity at 70% during the whole domestication period, simultaneously watering every 15 days after domestication and transplanting for 30 days, 0.2% water-soluble compound fertilizer (15:15:15), domesticating for three months, the domestication survival rate is 99.3%, pseudobulb expands, the formation rate of the "dragon egg" of the Cremastra appendiculata tissue culture seedling cut in one-step seedling multiplication culture is 12.8%, and the formation rate of the "dragon egg" of the Cremastra appendiculata tissue culture seedling cultured by one-step seedling culture is 69.4%.
Example 3
A method for inducing plants to sprout in one step by using pseudobulbs of rhododendron comprises the following steps:
1. selection and cleaning of explants: selecting Cremastra appendiculata, transplanting to indoor, irrigating root once every 3 days with 500 times of 75% (v/v) carbendazim solution, and irrigating root 5 times continuously; after the carbendazim is filled into roots, underground pseudobulbs are taken, overground stem leaves and surface root systems are removed, the pseudo bulbs are rinsed for 15min by using a saturated soap water solution, tap water is used for rinsing for 4 times, raised parts and depressed parts of the epidermis are scraped by using a knife, then 3 drops of Tween-80 are added, the mixture is shaken for 15min, and then tap water is used for showering for 80 min.
2. Explant disinfection: the cleaned explants were transferred to a clean bench, sterilized with 75% (v/v) alcohol for 25s, rinsed with sterile water for 3 times, sterilized with saturated bleaching powder solution for 20min, rinsed with sterile water for 4 times, sterilized with 0.5% benzalkonium bromide solution for 25min, rinsed with sterile water for 4 times, sterilized with 0.08% mercuric chloride solution for 7min, rinsed with sterile water for 8 times, the sterilization contamination rate was 7.1%, and the sterilization mortality rate was 1.1%.
3. And (3) induction culture of protocorms: the sterilized explants are cut into about 1.0cm after absorbing surface moisture with sterile paper3Having epidermal bits, inoculated in N6+2, 4-D0.5 mg/L +2-IP 1.2mg/L + BR 0.1mg/L + coconut milk 30g/L + active carbon 0.5g/L protocorm induction medium, culturing under the complete dark condition with the temperature of 25 +/-2 ℃ for the first 20 days, and culturing at the temperature of 25 +/-2 ℃ after 200 daysThe original bulb is cultured for 30 days in a light-dark alternative way under the conditions of 2 ℃, the illumination intensity is 1500Lux and the single-day illumination is 10 hours, and the induction rate of the original bulb is 70.1 percent.
4. One-step seedling multiplication culture: cutting the induced protocorm into 1.5cm long, inoculating to modified B5+ NAA 0.6mg/L + IBA 1.2mg/L + TDZ 0.8mg/L + BR 0.13mg/L + CH 70mg/L + coconut juice 50ml/L + activated carbon 0.5g/L, in the condition of 25 + -2 deg.C, illumination intensity 3000Lux, sunshine 12h, light-dark alternate culture for 60 days, differentiation rate is 99.7%, rooting rate is 95.9%, pseudobulb forming rate is 91.0%, proliferation coefficient is 15.4.
5. One-step seedling culture: taking out and domesticating the whole plant obtained by one-step seedling multiplication culture, cutting off the rest protocorm, cutting into 1.5cm long, and inoculating to the improved B5+ NAA 0.6mg/L + IBA 1.2mg/L + TDZ 0.8mg/L + BR 0.13mg/L + CH 70mg/L + coconut juice 50ml/L + activated carbon 0.5g/L, culturing alternately in dark and light conditions at 25 + -2 deg.C, illumination intensity 3000Lux and sunshine light 12h for 60 days, with differentiation rate of 100%, rooting rate of 95.4% and pseudobulb formation rate of 92.1%, (as shown in FIG. 3);
6. transplanting the rhododendron tissue culture seedlings obtained by one-step seedling formation into peat soil: perlite: in a matrix with coconut chaff at a ratio of 3:1:3, in a domestication environment under a greenhouse with a shading rate of 85%, keeping the air humidity at 100% for ten days before transplanting, then gradually reducing the air humidity, keeping the matrix humidity at about 70% during the whole domestication period, simultaneously pouring 0.2% water-soluble compound fertilizer (15:15:15) every 15 days after domestication and transplanting for 30 days, domesticating for three months, wherein the domestication survival rate is 99.3%, pseudobulb expands, the formation rate of dragon eggs of the Cremastra Appendiculata tissue culture seedlings after one-step seedling multiplication culture cutting is 14.5%, and the formation rate of dragon eggs of the Cremastra appendiculata tissue culture seedlings obtained by one-step seedling culture is 76.3%.
As can be seen from example 1, the protocorm induced by using pseudobulb as explant can be cultured in one step to obtain commercial seedlings for domestication by one step of seedling culture medium (as shown in FIG. 1). While the one-step seedling culture medium can generate a large amount of protocorms (as shown in figure 2) while performing differentiation and rooting, it can be seen from examples 2 and 3 that the protocorms can be further used for the further seedling culture (as shown in figure 3). Meanwhile, it can be seen from the above three examples that the formation rate of the cymbidium andraeanum 'dragon egg' can be increased when the protocorm is domesticated in the field along with the rooted seedling.
Finally, it should be noted that: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and it is intended to cover in the claims the invention as defined in the appended claims.
Claims (4)
1. A culture medium for inducing one-step seedling formation of a pseudobulb plant of Rhododendron simsii, the culture medium comprising a protocorm induction culture medium and a one-step seedling formation culture medium,
the formula of the protocorm induction culture medium is as follows: n is a radical of hydrogen6+2, 4-D0.3-0.5 mg/L +2-IP 1.0-1.2 mg/L + BR 0.05-0.1 mg/L + coconut milk 20-30 g/L + active carbon 0.5 g/L;
the formula of the one-step seedling culture medium is as follows: improvement B5+ NAA 0.5-0.6 mg/L + IBA 1.0-1.2 mg/L + TDZ 0.5-0.8 mg/L + BR 0.1-0.13 mg/L + CH 50-70 mg/L + coconut juice 40-50 ml/L + activated carbon 0.5 g/L.
2. The culture medium of claim 1, wherein the improvement B5The culture medium is original B5On the basis of the culture medium, the concentration of potassium nitrate is changed to 1500mg/L, the concentration of ammonium sulfate is changed to 205mg/L, the concentration of calcium chloride is changed to 125mg/L, the concentration of nicotinic acid is changed to 1.5mg/L, the concentration of pyridoxine hydrochloride is changed to 1.8mg/L, the concentration of zinc sulfate is changed to 3.0mg/L, and the other elements are not changed.
3. A method for inducing one-step shoot formation of a pseudobulb plant of azalea by using the medium of any one of claims 1 or 2, comprising the steps of:
(1) explant selection and cleaning: selecting and transplanting the azalea orchid indoors, irrigating the roots once every 3 days by using 500 times of 75% carbendazim solution, irrigating the roots for 4-5 times continuously, after irrigating the roots by using the carbendazim, taking underground pseudobulbs, removing overground stems, leaves and surface roots, rinsing for 10-15 min by using saturated soap water solution, rinsing for 3-4 times by using tap water, scraping the convex and concave parts of the epidermis by using a knife, adding 2-3 drops of Tween-80, shaking for 10-15 min, and rinsing for 60-80 min by using the tap water;
(2) explant disinfection: cleaning the explant, transferring the explant to a super clean workbench, disinfecting with 75% (v/v) alcohol for 20-25 s, rinsing with sterile water for 2-3 times, disinfecting with a saturated bleaching powder solution for 18-20 min, rinsing with sterile water for 3-4 times, disinfecting with a 0.5% neodell solution for 20-25 min, rinsing with sterile water for 3-4 times, disinfecting with a 0.08% (v/v) mercuric chloride solution for 6-7 min, and rinsing with sterile water for 6-8 times;
(3) and (3) induction culture of protocorms: the sterilized explants are cut into 1.0cm after absorbing surface water with sterile paper3Having epidermal pieces, inoculated in N6+2, 4-D0.3-0.5 mg/L +2-IP 1.0-1.2 mg/L + BR 0.05-0.1 mg/L + coconut milk 20-30 g/L + active carbon 0.5g/L protocorm induction medium, culturing for 20 days under the complete dark condition with the temperature of 25 +/-2 ℃, and after 20 days, culturing for 30 days under the conditions of 25 +/-2 ℃, the illumination intensity of 1000-2000 Lux and single day illumination for 10 hours in light and dark alternation;
(4) one-step seedling culture: inducing protocorm to cut into 1-1.5 cm, inoculating to modified B5+ NAA 0.5-0.6 mg/L + IBA 1.0-1.2 mg/L + TDZ 0.5-0.8 mg/L + BR 0.1-0.13 mg/L + CH 50-70 mg/L + coconut juice 40-50 ml/L + active carbon 0.5g/L, and culturing in a one-step seedling culture medium under the conditions of 25 +/-2 ℃, illumination intensity of 2000-3000 Lux and daily illumination for 12h for 60-80 days in a light-dark alternating manner.
4. The use of the method of claim 3 for inducing one-step plantlet formation in the breeding of Rhododendron simsii by using pseudobulbs of Rhododendron simsii.
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